乙草胺—Cu～（2+）复合污染条件下黑土农田生态系统微生物过程的研究
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摘要
目前在农药使用及研究过程中存在如下问题：多品种农药同时或交替使用造成复合污染具有普遍性；对复合污染土壤微生物生态效应的研究尚未引起足够重视；农药复合污染条件下土壤微生物种群结构变化及其适应复合污染条件的分子生态学机制研究尚缺乏认识。
采用传统毒性评价手段(微生物生物量、土壤生理指标)和现代分子生物学技术(16S rDNA指纹图谱分析)相结合的方法，研究了乙草胺和金属铜单一与复合污染对黑土的微生态胁迫，包括对黑土土壤微生物数量、植物生长有益菌、土壤脱氢酶活性和土壤呼吸强度的影响；同时，在建立土壤基因组提取及16S rDNA扩增方法的基础上，就黑土对乙草胺和铜单一与复合污染的分子生态响应进行了研究；对黑土中乙草胺抗性菌进行了筛选并对其降解能力进行研究，还对筛选到的降解乙草胺的一株菌株进行了16S rDNA序列分析。主要研究结果如下：
1．乙草胺-Cu~(2+)复合污染可明显促进土壤呼吸强度增强，促进率为14.10％；两者的抑菌作用和抑制土壤脱氢酶活性较其单因子均为最强，抑制率分别为93.15％(细菌)、89.68％(放线菌)、24.46％(真菌)、94.97％(自生固氮菌)、89.80％(硅酸盐细菌)、85.75％(矿化磷细菌)、35.03％(土壤脱氢酶活性)，表现为明显的加成效应。
2．采用优化成分的DNA裂解液提取DNA，并使用nest-PCR和变温退火手段扩增16S rDNA。
3．在环境污染的分子生态响应方面，长期施用农药土壤与未施农药清洁土壤存在明显不同，而与乙草胺-Cu~(2+)高剂量复合污染土壤在细菌群落结构上具有较高的相似性，相似系数达88％。在长期施用农药土壤中出现3条特异性带，并在乙草胺-Cu~(2+)高剂量复合污染时再次出现且亮度增强，而在单一污染及低剂量复合污染时均不出现。
4.从方法学角度分析，高剂量污染物对土壤微生物短期、急性暴露的模拟
培养在分子水平上能够反映污染物对环境微生态区系的长期影响和作用，可
以为污染物农田生态系统安全评价提供方法参考。
5.两种技术的结合能够更准确、更客观地判断外源污染物对微生物群落的
影响，也将是未来环境污染生态风险评价的趋势。
6.从污染土壤中筛选出可降解乙草胺的菌株524，其降解率可达62%。对
其165 rDNA进行序列测定，发现它与产酸克雷伯菌属(Klebsiellapneumoniae)
的多株菌序列同源性为99%，初步鉴定为产酸克雷伯菌属。
Joint toxicological effects of acetochlor and copper on indigenous bacterial communities in phaeozem were investigated by using the traditional toxicity assessment methods combined with the modern molecular biotechnology. The polyphasic approach combines (i) microbial enumeration with media, (ii) substrate-induced respiration(SIR), (iii) dehydrogenase activity, and (iv) denaturing gradient gel electrophoresis (DGGE) fingerprinting of total community DNA with bacterial primers. We had established and developed a simple and rapid protocol for genome extraction and 16S rDNA amplification from phaeozem. Meantime, one bacteria capable of utilizing acetochlor as sole carbon and energy sources was isolated from polluted phaeozem.
It was indicated that acetochlor and Cu2+ had a synergic inhibitory effect on the amount of culture-dependent viable microorganisms including bacteria (93.74%), actinomyces (93.0%), nitrogen-fixing bacteria (88.76%), silicate bacteria (96.47%) and phosphorus-mineralization bacteria (95.56%), as well as the activity of soil dehydrogenase (35.03%). The increase in SIR and the amount of fungi were significantly promoted by the two pollutants. Total DNA in soil was extracted using the optimized lysis buffer and 16S rDNA was amplified by nest-PCR and changing temperature condition. Similarity dendrograms showed that the bacterial community structures of the polluted soils and the clean soil without pesticide application were prominently different each other. Microbial community structures of phaeozem with long-term herbicides application were similar to those treated using higher concentration of acetochlor and Cu2+ simultaneously. The similarity
coefficient was up to 88%. There were three special bands in both.
The degradation rate of acetochlor by strain SZ4 was 62%. It was elementarily considered to be belonged to Klebsiella pneumonias, based on the sequence analyses of 16S rDNA.
采用传统毒性评价手段(微生物生物量、土壤生理指标)和现代分子生物学技术(16S rDNA指纹图谱分析)相结合的方法，研究了乙草胺和金属铜单一与复合污染对黑土的微生态胁迫，包括对黑土土壤微生物数量、植物生长有益菌、土壤脱氢酶活性和土壤呼吸强度的影响；同时，在建立土壤基因组提取及16S rDNA扩增方法的基础上，就黑土对乙草胺和铜单一与复合污染的分子生态响应进行了研究；对黑土中乙草胺抗性菌进行了筛选并对其降解能力进行研究，还对筛选到的降解乙草胺的一株菌株进行了16S rDNA序列分析。主要研究结果如下：
1．乙草胺-Cu~(2+)复合污染可明显促进土壤呼吸强度增强，促进率为14.10％；两者的抑菌作用和抑制土壤脱氢酶活性较其单因子均为最强，抑制率分别为93.15％(细菌)、89.68％(放线菌)、24.46％(真菌)、94.97％(自生固氮菌)、89.80％(硅酸盐细菌)、85.75％(矿化磷细菌)、35.03％(土壤脱氢酶活性)，表现为明显的加成效应。
2．采用优化成分的DNA裂解液提取DNA，并使用nest-PCR和变温退火手段扩增16S rDNA。
3．在环境污染的分子生态响应方面，长期施用农药土壤与未施农药清洁土壤存在明显不同，而与乙草胺-Cu~(2+)高剂量复合污染土壤在细菌群落结构上具有较高的相似性，相似系数达88％。在长期施用农药土壤中出现3条特异性带，并在乙草胺-Cu~(2+)高剂量复合污染时再次出现且亮度增强，而在单一污染及低剂量复合污染时均不出现。
4.从方法学角度分析，高剂量污染物对土壤微生物短期、急性暴露的模拟
培养在分子水平上能够反映污染物对环境微生态区系的长期影响和作用，可
以为污染物农田生态系统安全评价提供方法参考。
5.两种技术的结合能够更准确、更客观地判断外源污染物对微生物群落的
影响，也将是未来环境污染生态风险评价的趋势。
6.从污染土壤中筛选出可降解乙草胺的菌株524，其降解率可达62%。对
其165 rDNA进行序列测定，发现它与产酸克雷伯菌属(Klebsiellapneumoniae)
的多株菌序列同源性为99%，初步鉴定为产酸克雷伯菌属。
Joint toxicological effects of acetochlor and copper on indigenous bacterial communities in phaeozem were investigated by using the traditional toxicity assessment methods combined with the modern molecular biotechnology. The polyphasic approach combines (i) microbial enumeration with media, (ii) substrate-induced respiration(SIR), (iii) dehydrogenase activity, and (iv) denaturing gradient gel electrophoresis (DGGE) fingerprinting of total community DNA with bacterial primers. We had established and developed a simple and rapid protocol for genome extraction and 16S rDNA amplification from phaeozem. Meantime, one bacteria capable of utilizing acetochlor as sole carbon and energy sources was isolated from polluted phaeozem.
It was indicated that acetochlor and Cu2+ had a synergic inhibitory effect on the amount of culture-dependent viable microorganisms including bacteria (93.74%), actinomyces (93.0%), nitrogen-fixing bacteria (88.76%), silicate bacteria (96.47%) and phosphorus-mineralization bacteria (95.56%), as well as the activity of soil dehydrogenase (35.03%). The increase in SIR and the amount of fungi were significantly promoted by the two pollutants. Total DNA in soil was extracted using the optimized lysis buffer and 16S rDNA was amplified by nest-PCR and changing temperature condition. Similarity dendrograms showed that the bacterial community structures of the polluted soils and the clean soil without pesticide application were prominently different each other. Microbial community structures of phaeozem with long-term herbicides application were similar to those treated using higher concentration of acetochlor and Cu2+ simultaneously. The similarity
coefficient was up to 88%. There were three special bands in both.
The degradation rate of acetochlor by strain SZ4 was 62%. It was elementarily considered to be belonged to Klebsiella pneumonias, based on the sequence analyses of 16S rDNA.
引文
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